Staphylococcus aureus is a bacterial pathogen that is capable of colonizing a wide range of host tissues and causing infections such as endocarditis, wound infections, pneumonia, osteomyelitis and septic arthritis. The initial attachment and colonization on host tissues is thought to be the first crucial step in the disease development. Staphylococci produce a family of surface proteins named MSCRAMMs (Microbial Surface Component Recognizing Adhesive Matrix Molecules) that mediate adherence to extracellular matrix (ECM) proteins. Collagen is a major ECM protein and is the main component of tissues such as skin, bone, tendon, cartilage and teeth. It is conceivable that the S. aureus developed strategies to utilize collagen as a way to colonize host tissues.
Several studies have supported this notion. For example, S. aureus cells retrieved from infected joints were shown to be predominantly attached to the cartilage (1). Switalski et al. demonstrated that a collagen-binding MSCRAMM, CNA, was necessary and sufficient for S. aureus to attach to cartilage in vitro (2). Furthermore, CNA was shown to be a virulence factor in experimental septic arthritis (3). CNA+ strains showed dramatically increased virulence compared with the isogenic CNA− strains as evaluated by the clinical symptoms and histopathological patterns of the joints. No viable S. aureus cells were recovered from the joints of mice injected with the CNA− strains, while significant numbers of S. aureus cells were isolated from the joints of those injected with the CNA+ strains. In addition, vaccination with a recombinant fragment of CNA protected mice from septic death against intravenous challenge with S. aureus (4).
Other bacteria that have been reported to express collagen-adhesins include certain strains of Escherichia coli (5), Yersinia enterocolitica (6–8), Klebsiella pneumoniae (9), Streptococcus mutans (10,11), group A streptococci (12), Streptococcus gordonii (11,13), Enterococcus faecalis (14), Lactobacilli reuteri (15) and Lactobacilli crispatus (T. K. Korhonen, personal communication). The collagen-adhesin YadA was shown to contribute to the arthritogenicity of Y. enterocolitica in a rat model (16). Substitution of two histidine residues in YadA with alanine abrogated collagen binding and cell adherence, and caused reduced virulence in mice (17), suggesting that these functions of YadA were related. Mammalian proteins that interact with collagen include extracellular matrix proteins (e.g., fibronectin, decorin, thrombospondin and von Willebrand factor) and cell receptors (e.g., integrins α1β1 and α2β1). Their interactions mediate processes such as cell adhesion and migration, platelet aggregation, and tumor metastasis. Therefore, a deeper understanding of the interactions between collagen and the staphylococcal adhesin will not only contribute to our understanding of the molecular pathogenesis of staphylococcal infections but also provide insight of how collagen-binding proteins in general interact with this ligand.
Previous studies showed that CNA recognized triple helical collagen as well as some synthetic peptides that formed a collagen-like triple helix (18). CNA was found to contain typical elements of surface proteins of gram-positive bacteria. It consists of a signal peptide, a non-repetitive A domain, several repeats (B domains), followed by a cell-wall anchor region, a transmembrane segment and a short cytoplasmic tail. The A domain of CNA (CNA55) was found to be fully responsible for the collagen-binding activity of CNA (19). The minimum binding domain was localized to a 19 kDa fragment, CNA19 (formerly designated CBD (151–318)), within CNA55 (19). The crystal structure of CNA19 was solved (20). The structure was composed of two antiparallel β-sheets and two short α-helices. β-strands A, B, part of D, E and H form β-sheet I, and strands C, part of D, F, G, I and J form β-sheet II. A trench was observed in β-sheet I into which a triple helical collagen molecule fits as shown by docking experiments using theoretical collagen probes of [(Gly-Pro-Pro)4]3 or [(Gly-Pro-Hyp)4]3. Site-directed mutagenesis of some residues in the trench of CNA19 abolished collagen binding (Y175K, R189A, F191A, N193K, and Y233A) or caused reduced binding affinity (N223K and N278K) (20,21), indicating that the trench was essential for binding. Two truncates of CNA19 that had the N-terminal 30 amino acids or the C-terminal 22 amino acids removed respectively were generated. The C-terminal truncate, CBD (151–297), did not bind collagen, while the N-terminus truncate was insoluble. The N-terminal truncate contains all but 6 of the residues in the trench and the C-terminal truncate contains all the residues, suggesting that the intact CNA19 molecule is important in presenting the trench in an active collagen-binding conformation. Structural analysis on the I domains of integrins α1β1 and α2β1, the collagen binding domains, also indicated the presence of trench-like structures (22–24). Mutations of some residues along the wall of the trench in the I domain of α2β1 affected collagen binding (25–28). Thus, it seems that using a trench structure as the site of interaction for collagen may be a general theme.
Recently, a collagen-adhesin (ACE) of Enterococcus faecalis was identified (14). ACE has similar domain organization as CNA, a signal peptide sequence, a collagen-binding region (A) followed by repeated segments (B repeats), and elements required for surface display. The A domain of ACE shares sequence similarity with the A domain of CNA in the corresponding segments and these parts of the two proteins appear to be structurally related as determined by far-UV circular dichroism analysis of the ACE A domain (14). This raised the possibility that there may also be a trench structure in the ACE A domain and the trench may be the binding site for collagen.
However, with regard to Staphylococcal bacteria, it has still remained a problem to identify and utilize the information concerning the collagen binding site so as to develop methods of more efficiently preventing and/or treating staph infections. In addition, it has still been a goal of the study of the collagen binding activity of to develop methods and compositions which could be useful in treating or preventing infections from more than one type of staph infection. Finally, it is even further desirable in the field of infectious diseases to development compositions which are particularly successful not only in preventing staph infection, but in treating existing infection by displacing proteins already bound at sites on the extracellular matrix in cells of a human or animal patient.